Postharvest mycoflora and genetic diversity of fungi causing anthracnose on mango fruits in Western India

Introduction

Mango (Mangifera indica L.) is recognized as one of the most economically important tropical fruit crops grown globally, appreciated for its nutritional value and potential for commercial export. In 2023, worldwide mango production surpassed 57 million metric tonnes, with India accounting for roughly 41% of the overall supply (FAO, 2024). The fruit is commonly enjoyed fresh and is also processed into various value-added products such as pulp, puree, beverages, frozen slices, and dried chips. Nevertheless, despite its significant market demand, mango is particularly susceptible to postharvest spoilage, leading to considerable quantitative and qualitative losses during handling, storage, transportation, and export. Annual postharvest losses for mango are estimated to be between 25% and 45% in tropical supply chains, with fungal diseases being a major contributing factor [12] .Among postharvest diseases, anthracnose is acknowledged as the most severe, widespread, and economically detrimental disease impacting mango fruits. This disease is mainly caused by species within the Colletotrichum gloeosporioides species complex; however, several phylogenetically distinct yet morphologically similar species have been reported, including C. siamense, C. asianum, and C. fructicola [14] . The pathogen employs a distinctive quiescent infection strategy, infecting flowers and immature fruits during their early development and remaining dormant until the fruit ripens, at which point host physiological changes and environmental humidity activate necrotrophic behavior [3,8] . This latent characteristic renders preharvest detection challenging and significantly complicates the management of postharvest diseases.In addition to Colletotrichum, mango fruits are also susceptible to stem-end rot pathogens, especially species of Lasiodiplodia (Botryosphaeriaceae), with L. theobromae and L. pseudotheobromae being the most prevalent [11,13] . Lasiodiplodia infections generally begin in the pedicel and vascular tissues, remaining asymptomatic until the postharvest phase, particularly in conditions of high humidity and poor temperature regulation [6] . The combination of Colletotrichum anthracnose and Lasiodiplodia stem-end rot leads to swift postharvest fruit deterioration, tissue maceration, and significant decreases in market value. Importantly, recent findings suggest that these pathogens may coexist or interact synergistically, thereby improving the efficiency of host tissue colonization [9] . Despite the significance of these pathogens, morphological diagnosis is still inadequate for precise species identification. Colletotrichum and Lasiodiplodia spp. often exhibit phenotypic plasticity that is affected by factors such as medium, temperature, and light, which results in frequent misidentification in standard plant pathology laboratories [5,14] . As a result, molecular techniques especially the sequencing of the Internal Transcribed Spacer (ITS) region have become the norm for fungal species delimitation, phylogenetic analysis, and disease monitoring [16] . ITS based barcoding facilitates the differentiation of morphologically cryptic species and uncovers connections between pathogen prevalence and regional agroecological selection pressures. Nevertheless, within the framework of the Western Indian mango belt (Konkan-Nashik-Paithan), there is a scarcity of systematic surveys on postharvest mycoflora. The region's warm and humid maritime climate, prolonged monsoon season, and dense orchard canopy structure foster conditions that are conducive to the persistence and dispersal of fungal inoculum. Additionally, high-value export cultivars such as Alphonso, Kesar, Ratna, and Sonpari are recognized for their varying levels of susceptibility to anthracnose and stem-end rot; however, the specific pathogen associations for each cultivar remain inadequately understood in this area. Consequently, this study aims to fill a significant knowledge gap by integrating morphological characterization of postharvest fungal isolates, ITS-based molecular identification and phylogenetic analysis, as well as an assessment of isolate frequency and diversity across 34 mango cultivars. This approach seeks to identify the dominant pathogens, genetic variation, and ecological interactions that contribute to postharvest decay in mango fruits within Western India. By merging fungal diversity profiling with phylogenetic and nucleotide composition analysis, this research offers fresh insights into hostpathogen distribution patterns, regional disease pressures, and the evolutionary dynamics of Colletotrichum and Lasiodiplodia populations that impact mango supply chains. The findings are designed to aid in the formulation of integrated postharvest management systems, which include molecular diagnostics, cold-chain optimization, and biocontrol-based strategies for disease mitigation, all in alignment with sustainable horticultural production and export quality standards.

Materials and Methods

Sample Collection

During the 2023 harvesting season, mango fruits displaying typical signs of anthracnose, stem-end rot, or soft rot were gathered from commercial orchards and markets in the Konkan, Nashik, and Paithan areas of Western India. A total of 54 fruit samples from 34 different cultivars were included (Table 1). The geographic coordinates for each sampling location were documented using GPS technology. The fruits were placed in sterile paper bags, transported to the laboratory, and processed within a 24-hour timeframe.

Isolation of Fungi

Symptomatic peel and pulp tissues (5-7 mm) from the edges of lesions (Figure 1) were subjected to surface sterilization (70% ethanol for 30 seconds, 2% NaOCl for 90 seconds, followed by a rinse with sterile water), then blotted dry and placed onto Potato Dextrose Agar (PDA). The plates were incubated at a temperature of 28±2 °C for a duration of 5-7 days. The colonies that emerged were subsequently purified. Through single spore isolation and maintained on PDA slants at 4 °C for further analysis.

Morphological Characterization

The morphology of the colony, its pigmentation, growth patterns, and sporulation were documented following a 7day incubation period. Conidial structures were analyzed using a light microscope, with dimensions measured from at least 50 measurements for each isolate. Identification was conducted in accordance with the diagnostic keys established by [4,15] . The PCR products were confirmed on 1.2% agarose gels, purified, and sequenced in both directions using commercial Sanger sequencing services.

Sequence Analysis and Species Identification

Consensus sequences were compiled and contrasted with reference sequences in NCBI GenBank utilizing BLASTn for species-level identification. GC content, base composition, and pairwise sequence divergence were determined using MEGA X. Phylogenetic analysis utilized the Neighbor-Joining method with 1,000 bootstrap replicates to deduce evolutionary relationships among isolates.

Frequency and Diversity Assessment

The relative frequency of each fungal genus and species was determined by calculating the total representation of isolates across various cultivars. The diversity indices and clade groupings were analyzed in relation to the prevalence of postharvest infections and their ecological associations within the storage and handling processes.

Results and Discussion Morphological Characterization of Postharvest Fungal Isolates

A total of 54 fungal isolates were obtained from symptomatic mango fruits, which represented 34 different cultivars throughout the Konkan region. The isolates displayed unique colony morphologies on PDA. Colletotrichum spp. generated colonies that were grey to orange-pink and velvety, characterized by cylindrical, hyaline conidia. Conversely, Lasiodiplodia isolates developed dark grey to olivaceous, cottony colonies, featuring thick-walled, ellipsoidal conidia that were initially hyaline but darkened upon maturation. Opportunistic isolates of Aspergillus, Alternaria, and Nigrospora were easily identified by their conidial sporulation structures. The morphological characteristics provided support for preliminary classification at the genus level as shown in Table 1. Pairwise distance estimates revealed low intra-species divergence inter-genus divergence (greater than 0.45). (<0.05) and high inter-genus divergence (>0.45) as shown in Table 5 and Figure 3.

Pathogen Frequency and Distribution

Analysis of pathogen frequency revealed that Colletotrichum and Lasiodiplodia each represented 33.3% of all isolates. The remaining approximately 30% was made up of Nigrospora, Aspergillus, and Alternaria combined. The distribution patterns of pathogens were uniform across different cultivars, indicating a stable inoculum pressure throughout the region rather than a preference for infection specific to any cultivar. No unique associations between cultivars and pathogens were identified as shown in Table 6.

Discussion

The current research illustrates that the postharvest deterioration of mango fruits in Western India is primarily caused by species from the Colletotrichum gloeosporioides complex and the Lasiodiplodia lineage of Botryosphaeriaceae. The prevalence of Colletotrichum gloeosporioides and C. siamense in anthracnose lesions is consistent with global findings that identify the C. gloeosporioides species complex as the main contributor to latent infections on the surfaces of mango fruits (Freeman et al., 2018; Sharma et al., 2020; Lima et al., 2023) [14,10] . These pathogens infiltrate during the early stages of fruit development but remain dormant until physiological changes induced by ripening trigger fungal activation (Prusky, et al., 2019) [12] . The transition from a quiescent to a necrotrophic state poses significant challenges in the warm, humid postharvest conditions typical of the Konkan coastal region, facilitating the rapid appearance of decay symptoms during marketing and storage. In a similar vein, the frequent occurrence of Lasiodiplodia theobromae and L. pseudotheobromae aligns with the growing acknowledgment of stem-end rot pathogens as significant postharvest risks throughout tropical mango supply chains (Meena et al., 2018; Dofuor et al., 2023; Sales et al., 2022) [11,7,13] . These fungi invade pedicel tissues and remain asymptomatic until the postharvest storage phase, especially under conditions of moisture stress and ethylene-induced softening. Importantly, Lasiodiplodia species thrive in the absence of adequate cold-chain systems, which is a persistent challenge in decentralized fruit markets and rural aggregation centers across India (Al-Sadi et al., 2021; Ciofini et al., 2022) [2,6] .The identification of opportunistic fungi such as Nigrospora sphaerica, Alternaria burnsii, and Aspergillus niger indicates the existence of a secondary necrotrophic complex that can colonize senescent or damaged tissues. Their simultaneous presence with Colletotrichum and Lasiodiplodia reinforces a successional decay model, wherein primary pathogens penetrate the initial layers of host defense, modifying tissue chemistry and allowing saprophytic fungi to thrive (Hyde et al., 2014) [9] . These interactions among microbial consortia exacerbate the loss of fruit firmness, degradation of flavor, and the swift progression of brown rot during transport. The combination of morphological and ITS-based identification in this research highlights the inadequacies of relying solely on morphology, especially within the Colletotrichum gloeosporioides complex, where similar conidial forms frequently result in taxonomic misinterpretation (Cannon et al., 2012) [5] . The recorded GC content range (45.8-60.2%) further illustrates significant sequence variability among anthracnose pathogen clades, corroborating earlier findings of cryptic species divergence and host-adaptive evolution. Phylogenetic analysis distinctly separated the lineages of Colletotrichum and Lasiodiplodia, with strong bootstrap support, confirming ITS as a dependable first-tier diagnostic marker for monitoring postharvest pathogens. Significantly, the patterns of pathogen prevalence were uniform across all mango cultivars, including the commercially prevalent Alphonso, Ratna, Kesar, and Sonpari. This indicates that resistance specific to cultivars is not prominently exhibited at the postharvest phase, aligning with previous research that suggests resistance traits manifested during on-tree growth may not effectively inhibit the reactivation of latent postharvest infections (Arauz, 2000) [3] . This further underscores the importance of interventions in postharvest handling, which include: hot-water treatment or fungicidal dips immediately following harvest, optimized cold-chain logistics to delay quiescent activation, sanitized harvesting and storage equipment to minimize the spread of inoculum, biocontrol agents such as Trichoderma harzianum or Pseudomonas fluorescens, and plant-derived antifungal volatiles (for instance, lemongrass and neem oils) that can inhibit Colletotrichum by 70-90% in vitro. In light of the global trend towards reduced reliance on fungicides due to regulations on residues and the emergence of pathogen resistance (Abd El-Baky et al., 2021) [1] , it is crucial to integrate molecular diagnostics with bio-based antifungal approaches to ensure sustainable protection of the mango supply chain. In summary, the results of this study offer definitive molecular evidence of pathogen diversity, distribution, and co-occurrence dynamics that influence the postharvest stability of mangoes in Western India. These findings establish a vital basis for the development of predictive disease monitoring systems, the enhancement of export quality standards, and the direction of future breeding and biocontrol strategies aimed at addressing the anthracnose-stem-end rot pathogen complex.

Conclusion

This research illustrates that the decay of mango fruits after harvest in Western India is mainly attributed to species within the Colletotrichum gloeosporioides complex and Lasiodiplodia spp., as evidenced by both morphological characteristics and ITS-based phylogenetic analysis. The simultaneous presence of these pathogens, along with opportunistic necrotrophs such as Alternaria, Nigrospora, and Aspergillus, suggests a collaborative decay mechanism that hastens the deterioration of fruit during storage and transportation. The consistent prevalence of pathogens across different cultivars indicates that the severity of postharvest diseases is more significantly affected by environmental factors and handling practices than by the natural resistance of the varieties themselves. These results underscore the necessity of incorporating molecular diagnostics, swift postharvest interventions, optimization of cold-chain logistics, and bio-based antifungal methods to mitigate economic losses and improve the quality of fruit supply chains for export in tropical areas.

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